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For dozens of years microsurgical techniques have been successfully applied in various procedural areas mainly in ophthalmology, laryngology, female infertility treatment, urology, plastic surgery and reconstruction as well as hand surgery. Courses enabling acquisition of basic qualifications within the microsurgical techniques area are the starting point for such activity, there is however an urgent need for further training as it goes without saying that without adequate training one may quickly forget all the experience gained during such trainings. Our department have been using foetal placenta obtained from maternity ward (following a written consent from a parturient) to carry out microsurgical trainings for 13 months now.
Isoelectric focusing of homogenous arylsulfatase B from human placenta pointed to the presence of enzymatically active and inactive forms of high pi (pH 9-8) and of lower pi (pH 6.5-5.5). Glycan chain analysis performed with the use of a Glycan Differentiation Kit showed that basic forms of arylsulfatase B from human placenta contained mostly high mannose/hybrid type glycans, with 6-O-L-fucose bound to the innermost AT-acetylglucosamine residue, whereas acidic forms of the enzyme contained complex type glycans containing fucose and sialic acid. However, the latter forms constitute a small percentage of the total carbohydrate component. Lectin affinity chromatography of the native enzyme confirmed the presence of a core fucose and a sialic acid.
Such a large organ as human placenta could be potentially a very effi - cient source of stem cells. In addition, taking of this organ is not invasive and controversial process. Isolation of cells from diff erent layers of the placenta is divided into several stages. From fetal part of human placenta hAEC (human amniotic epithelial cells), hAMSC (human amniotic mesenchymal stromal cells), hCMSC (human chorionic mesenchymal stromal cells) and hCTC (human chorionic trophoblastic cells) can be isolated. Among these cells are present stem cells, which are characterized by pluripotency. Stem cells derived from placenta express markers specifi c for embryonic stem cells (SSEA-3, SSEA-4, TRA1-60, TRA1-81), and they have the ability to diff erentiate towards cells representing three germ layers: mesoderm, endoderm and ectoderm, for example: adipocytes, osteoblasts, chondrocytes, endotheliocytes, skeletal muscle cells, cardiomyocytes, neurons, glial cells, pancreatic cells and hepatocytes. Cells isolated from human placenta are also characterized by low immunogenicity and tumorigenicity after implantation to the animals. Placenta-derived stem cells seem to be valuable population of cells for regenerative medicine and in a treatment of terminal diseases.
Potencjalnie, tak duży narząd jak łożysko ludzkie powinien być wydajnym źródłem komórek macierzystych. Pobieranie łożyska nie budzi kontrowersji. Izolacja komórek z poszczególnych warstw tego narządu jest procesem kilkuetapowym. Z części płodowej łożyska ludzkiego wyizolowano: komórki nabłonka owodni (hAEC), mezenchymalne komórki podścieliska owodni (hAMSC), mezenchymalne komórki podścieliska kosmówki (hCMSC) oraz komórki trofoblastu kosmówki (hCTC). Wśród tych komórek wykazano obecność komórek macierzystych posiadających cechy pluripotencjalności. Pochodzące z łożyska komórki macierzyste wykazują ekspresję markerów specyfi cznych dla embrionalnych komórek macierzystych (SSEA-3, SSEA-4, TRA1-60, TRA1-81), a także mają zdolność do różnicowania się w kierunku komórek reprezentujących trzy listki zarodkowe: mezodermę, endodermę i ektodermę, np. do adipocytów, osteoblastów, chondrocytów, endoteliocytów, komórek mięśnia szkieletowego, kardiomiocytów, neuronów, komórek glejowych, komórek trzustki i hepatocytów. Komórki izolowane z łożyska ludzkiego charakteryzują się niską immunogennością i nie wykazują tendencji do transformacji nowotworowej po przeszczepieniu zwierzętom doświadczalnym. Komórki macierzyste pochodzące z łożyska ludzkiego wydają się wartościową populacją komórek dla medycyny regeneracyjnej oraz w leczeniu wielu chorób, do tej pory nieuleczalnych.
The effects of Mg2+ and bicarbonate on the kinetics of ITP hydrolysis by soluble ATPase (Fi) from human placental mitochondria were studied. Increasing amounts of Mg2+ at fixed ITP concentration, caused a marked activation of Fi followed by inhibition at higher Mg2+ concentration. The appropriate substrate for the mitochondrial Fi seems to be the MglTP complex as almost no ITP was hydrolysed in the absence of magnesium. Mg2+ behaved as a competitive inhibitor towards the MglTP complex. In this respect the human placental enzyme differ from that from other sources such as yeast, beef liver or rat liver. The linearity of the plot presenting competitive inhibition by free Mg2+ of MglTP hydrolysis (in the presence of activating bicarbonate anion) suggests that both Mg2+ and MglTP bind to the same catalytic site (Km(MgITP) = 0.46 mM, Ki(Mg) = 4 mM). When bicarbonate was absent in the ITPase assay, placental Fi exhibited apparent negative cooperativity in the presence of 5 mM Mg2+, just as it did with MgATP as a substrate under similar conditions. Bicarbonate ions eliminated the negative cooperativity with respect to ITP (as the Hill coefficient of 0.46 was brought to approx. 1), and thus limited inhibition by free Mg2+. The results presented suggest that the concentration of free magnesium ions may be an important regulatory factor of the human placental Fi activity.
Morphochemical tests of placentae and clinical examinations of babies born-at-term with intrauterine hypotrophy and of normal body weight were conducted. A decreased activity of cytochrome c oxidase in syncytiotrophoblast, cytotrophoblast extravillous, amniotic epithelium as well as in decidual cells was observed. In all placenta samples we found a diminished percentage of intesvillous space, disordered maturing of villi, lower epithelial plates and striking loss of cytochrome c oxidase activity. It was concluded that diminished metabolic efficiency of placentae is the decisivee risk factor for hypotrophy in chemically polluted areas.
Mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after Parkinson’s disease. MSCs have significant advantages over other stem cell types, and greater potential for immediate clinical application. The aim of this study was to investigate whether MSCs from the human placenta could be induced to differentiate into dopaminergic cells. MSCs from the human placenta were isolated by digestion and density gradient fractionation, and their cell surface glycoproteins were analyzed by flow cytometry. These MSCs were cultured under conditions promoting differetiation into adipocytes and osteoblasts. Using a cocktail that includes basic fibroblast growth factor (bFGF), all trans retinoic acid (RA), ascorbic acid (AA) and 3-isobutyl-1-methylxanthine (IBMX), the MSCs were induced in vitro to become dopamine (DA) neurons. Then, the expression of the mRNA for the Nestin and tyrosine hydroxylase (TH) genes was assayed via RT-PCR. The expression of the Nestin, dopamine transporter (DAT), neuronal nuclear protein (NeuN) and TH proteins was determined via immunofluorescence. The synthesized and secreted DA was determined via ELISA. We found that MSCs from the human placenta exhibited a fibroblastoid morphology. Flow cytometric analyses showed that the MSCs were positive for CD44 and CD29, and negative for CD34, CD45, CD106 and HLA-DR. Moreover, they could be induced into adipocytes and osteocytes. When the MSCs were induced with bFGF, RA, AA and IBMX, they showed a change in morphology to that of neuronal-like cells. The induced cells expressed Nestin and TH mRNA, and the Nestin, DAT, NeuN and TH proteins, and synthesized and secreted DA. Our results suggest that MSCs from the human placenta have the ability to differentiate into dopaminergic cells.
Our study was designed to establish whether air pollution in urbanized industrial centers of southern Poland affects the process of glycosylation in a full-term human placenta. This process of glycosylation was analyzed by the quantitative determination of the binding of WGA and LCA lectins to placental villi. The study was performed on human placentas collected in 1990-91 and 2000-01 in regions of southern Poland differing in their degree of environmental pollution: the highly polluted areas of Upper Silesia and Cracow agglomeration. The Bieszczady area with low pollution was considered the control. The concentrations of nitrogen and sulfur oxides and the concentration of aerosols were used as markers of the degree of air pollution. The direct immunofluorescence reaction of the placenta tissues with fluorescein-labeled (FITC) lectins was used. The staining of the placenta tissues was examined under a fluorescence microscope linked to an analysis system. A microdensytometric method was used to assay the amount of tissue-bound lectins. The results showed no significant effect of the three main air pollutants in the study areas in southern Poland, i.e. nitrogen and sulfur oxides and high level of aerosols, on the structure of WGAand LCA-specific glycoconjugates in human placenta. However, the marked quantitative changes in the degree of lectin binding to placental cellular structures were noted within the last 10-year period in all studied regions.
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